The delivery of newly-synthesized membrane and secretory proteins via fast axonal transport to prescribed intracellular destinations is an absolute requirement for the maintenance of neuronal structure and function. The long term objective of the research is to understand the molecular recognition events that underlie sorting, routing and delivery of these fast-transported proteins. Evidence from non-neural systems indicate that regions of primary structure as well as co-translational and post-translational modifications are all candidates for intracellular sorting and destinations signals. In the current proposal two major approaches are designed to examine potential molecular destination markers of fast-transported proteins. First, a unique sub-population of the fast-transported proteins, identified during the previous grant period as containing sulfated tyrosine residues, will be studied to determine (i) effects of inhibiting sulfation on subsequent transport, (ii) the distribution of fast-transported "sulfoproteins" among different neuronal types, (iii) whether tyrosine sulfate earmarks proteins for secretion, and (iv) whether sulfation can occur in nerve terminals as well as in the Golgi apparatus. Second, immunological probes will be used to examine synthesis and early stages of fast transport of membrane and secretory proteins. Using antisera available to known fast-transported proteins, as well as antisera to be raised against fast-transported proteins characterized on two-dimensional gels, experiments will examine (i) if secretory and membrane proteins are synthesized on membrane-bound and/or free polysomes and (ii) whether the newly-synthesized products contain transient or long-lived amino-terminal signal sequences. Antisera will also be used to (iii) compare the intrasomal routing and kinetics of fast-transported secretory and membrane proteins. The analysis of individual fast-transported proteins will be completed by (iv) comparing how proteins are routed within the axon toward respective destinations. The identification of sub-populations of proteins that are subject to similar synthesis or routing, and/or transport will provide a further understanding of how fast-transported proteins reach prescribed destinations: how the complex organization of nerve cells is maintained.